Gingival crevicular fluid levels of apelin correlates with clinical periodontal diagnosis.

OBJECTIVES: Limitations of clinical periodontal measurements have led to the search for reliable biomarkers that can be used in diagnosis and monitoring of periodontal diseases. Considering the relationship of adipokines with periodontal disease, diabetes, and obesity, apelin may be a biomarker for periodontal diseases due to its modulating effects on inflammation. The present study was conducted to determine gingival crevicular fluid (GCF) apelin levels in systemically healthy individuals and to evaluate the potential of apelin as a biomarker for periodontal diagnosis. MATERIALS AND METHODS: Ten individuals with clinically healthy periodontal tissues, 10 patients diagnosed with gingivitis, and 10 patients with periodontitis were included in the present study. Whole mouth clinical periodontal measurements were recorded and GCF samples were obtained from the buccal approximal regions of single-rooted teeth with features that would represent clinical periodontal diagnosis. Apelin level in the samples was determined by ELISA. Clinical and biochemical findings were statistically analyzed. Possible relationship between the variables was evaluated with Pearson correlation analysis. RESULTS: Apelin level in the gingivitis group was higher than that in the clinically healthy group (p = 0.000) and lower than that in the periodontitis group (p = 0.000). A positive correlation was found between GCF apelin concentration and plaque score, bleeding on probing, and probing depth (p = 0.000). CONCLUSIONS: Within the limits of this study, it can be suggested that GCF apelin concentration may be a biomarker that can distinguish between healthy periodontal tissues, gingivitis, and periodontitis patients. CLINICAL RELEVANCE: Apelin concentration in the gingival crevicular fluid may aid in the diagnosis of periodontal disease.

Influence of Occlusal Hypofunction on Alveolar Bone Healing in Rats.

The aim of this in vivo study was to investigate the effect of occlusal hypofunction on alveolar bone healing in the absence or presence of an enamel matrix derivative (EMD). A standardized fenestration defect over the root of the mandibular first molar in 15 Wistar rats was created. Occlusal hypofunction was induced by extraction of the antagonist. Regenerative therapy was performed by applying EMD to the fenestration defect. The following three groups were established: (a) normal occlusion without EMD treatment, (b) occlusal hypofunction without EMD treatment, and (c) occlusal hypofunction with EMD treatment. After four weeks, all animals were sacrificed, and histological (hematoxylin and eosin, tartrate-resistant acid phosphatase) as well as immunohistochemical analyses (periostin, osteopontin, osteocalcin) were performed. The occlusal hypofunction group showed delayed bone regeneration compared to the group with normal occlusion. The application of EMD could partially, but not completely, compensate for the inhibitory effects of occlusal hypofunction on bone healing, as evidenced by hematoxylin and eosin and immunohistochemistry for the aforementioned molecules. Our results suggest that normal occlusal loading, but not occlusal hypofunction, is beneficial to alveolar bone healing. Adequate occlusal loading appears to be as advantageous for alveolar bone healing as the regenerative potential of EMD.

Apelin Enhances the Effects of Fusobacterium nucleatum on Periodontal Ligament Cells In Vitro.

This study aimed to explore effects of Fusobacterium nucleatum with or without apelin on periodontal ligament (PDL) cells to better understand pathomechanistic links between periodontitis and obesity. First, the actions of F. nucleatum on COX2, CCL2, and MMP1 expressions were assessed. Subsequently, PDL cells were incubated with F. nucleatum in the presence and absence of apelin to study the modulatory effects of this adipokine on molecules related to inflammation and hard and soft tissue turnover. Regulation of apelin and its receptor (APJ) by F. nucleatum was also studied. F. nucleatum resulted in elevated COX2, CCL2, and MMP1 expressions in a dose- and time-dependent manner. Combination of F. nucleatum and apelin led to the highest (p < 0.05) expression levels of COX2, CCL2, CXCL8, TNF-α, and MMP1 at 48 h. The effects of F. nucleatum and/or apelin on CCL2 and MMP1 were MEK1/2- and partially NF-κB-dependent. The combined effects of F. nucleatum and apelin on CCL2 and MMP1 were also observed at protein level. Moreover, F. nucleatum downregulated (p < 0.05) the apelin and APJ expressions. In conclusion, obesity could contribute to periodontitis through apelin. The local production of apelin/APJ in PDL cells also suggests a role of these molecules in the pathogenesis of periodontitis.

Obesity Modifies the Proteomic Profile of the Periodontal Ligament.

This study aimed to assess the obesity effects on the proteomic profile of the periodontal ligament of rats submitted to obesity induction by a high-fat diet. Eight Holtzman rats were divided into control (n = 3) and obese (n = 5) groups. The maxillae were histologically processed for laser capture microdissection of the periodontal ligament of the first maxillary molars. Peptide mixtures were analyzed by LC-MS/MS. A total of 1379 proteins were identified in all groups. Among them, 335 (24.30%) were exclusively detected in the obese group, while 129 (9.35%) proteins were uniquely found in the control group. Out of the 110 (7.98%) differentially abundant proteins, 10 were more abundant and 100 had decreased abundance in the obese group. A gene ontology analysis showed some proteins related to obesity in the "extracellular exosome" term among differentially identified proteins in the gene ontology cellular component terms Prelp, Sec13, and Sod2. These three proteins were upregulated in the obese group (p < 0.05), as shown by proteomic and immunohistochemistry analyses. In summary, our study presents novel evidence that the proteomic profile of the periodontal ligament is altered in experimental obesity induction, providing a list of differentially abundant proteins associated with obesity, which indicates that the periodontal ligament is responsive to obesity.

Obesity influences the proteome of periodontal ligament tissues following periodontitis induction in rats.

BACKGROUND AND OBJECTIVES: Many studies have been conducted to better understand the molecular mechanism involved with periodontitis progression. There has been growing interest in the potential impact of obesity on periodontitis onset and progression, but the mechanisms involved remain to be elucidated. The present study was designed to determine the impact of obesity on experimentally induced periodontitis in rats and identify novel pathways involved. METHODS: Sixteen Holtzman rats were distributed into two groups (n = 8): ligature-induced periodontitis (P) and obesity plus ligature-induced periodontitis (OP). Obesity was induced by a high-fat diet for 70 days, whereas periodontitis was induced for 20 days, with a cotton thread placed around the upper first molars bilaterally. Alveolar bone loss was measured by microtomographic analysis and histologically by histometry on the hemimaxillae. The protein composition of the periodontal ligament was evaluated by proteomic analysis. RESULTS: Data analysis (body weight, adipose tissue weight, and blood test) confirmed obesity induction, whereas bone loss was confirmed by micro-CT and histologic analyses. Proteome analysis from the periodontal ligament tissues (PDL) identified 819 proteins, 53 exclusive to the P group, 28 exclusive to the OP group, and 738 commonly expressed. Validation was performed by immunohistochemistry for selected proteins (spondin1, vinculin, and TRAP). CONCLUSION: Histologically, it was found that obesity did not significantly affect bone loss resulting from periodontitis. However, the present study's findings indicated that obesity affects the proteome of PDL submitted to experimental periodontitis, allowing for identifying potential targets for personalized approaches.

Cellular effects of glycine and trehalose air-polishing powders on human gingival fibroblasts in vitro.

OBJECTIVES: Air-polishing has been used in the treatment of periodontitis and gingivitis for years. The introduction of low-abrasive powders has enabled the use of air-polishing devices for subgingival therapy. Within the last decade, a wide range of different low-abrasive powders for subgingival use has been established. In this study, the effects of a glycine powder and a trehalose powder on human gingival fibroblasts (HGF) were investigated. METHODS: HGF were derived from three systemically and periodontally healthy donors. After 24 h and 48 h of incubation time, mRNA levels, and after 48 h, protein levels of TNFα, IL-8, CCL2, and VEGF were determined. In addition, NF-κB p65 nuclear translocation and in vitro wound healing were assessed. Statistical analysis was performed by ANOVA and post hoc Dunnett's and Tukey's tests (p < 0.05). RESULTS: Glycine powder significantly increased the expression of proinflammatory genes and showed exploitation of the NF-κB pathway, albeit trehalose powder hardly interfered with cell function and did not trigger the NF-κB pathway. In contrast to trehalose, glycine showed a significant inhibitory effect on the in vitro wound healing rate. CONCLUSION: Subgingivally applicable powders for air-polishing devices can regulate cell viability and proliferation as well as cytokine expression. Our in vitro study suggests that the above powders may influence HGF via direct cell effects. Trehalose appears to be relatively inert compared to glycine powder. CLINICAL RELEVANCE: With the limitations of an in vitro design, our study suggests that in terms of cell response, trehalose-based air-polishing powders show a reduced effect on inflammation.

Interaction of periodontitis and orthodontic tooth movement-an in vitro and in vivo study.

OBJECTIVES: The aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2). MATERIALS AND METHODS: The effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA. RESULTS: Periodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro. CONCLUSIONS: Our study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process. CLINICAL RELEVANCE: Orthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

Impact of Leptin on the Expression Profile of Macrophages during Mechanical Strain In Vitro.

Childhood obesity is a growing problem in industrial societies and associated with increased leptin levels in serum and salvia. Orthodontic treatment provokes pressure and tension zones within the periodontal ligament, where, in addition to fibroblasts, macrophages are exposed to these mechanical loadings. Given the increasing number of orthodontic patients with these conditions, insights into the effects of elevated leptin levels on the expression profile of macrophages during mechanical strain are of clinical interest. Therefore, the aim of this in vitro study was to assess the influence of leptin on the expression profile of macrophages during simulated orthodontic treatment. RAW264.7 macrophages were incubated with leptin and lipopolysaccharides (LPS) from Porphyromonas gingivalis (P. gingivalis) or with leptin and different types of mechanical strain (tensile, compressive strain). Expression of inflammatory mediators including tumor necrosis factor (TNF), Interleukin-1-B (IL1B), IL6, and prostaglandin endoperoxide synthase (PTGS2) was assessed by RT-qPCR, ELISAs, and immunoblot. Without additional mechanical loading, leptin increased Tnf, Il1b, Il6, and Ptgs2 mRNA in RAW264.7 macrophages by itself and after stimulation with LPS. However, in combination with tensile or compressive strain, leptin reduced the expression and secretion of these inflammatory factors. By itself and in combination with LPS from P. gingivalis, leptin has a pro-inflammatory effect. Both tensile and compressive strain lead to increased expression of inflammatory genes. In contrast to its effect under control conditions or after LPS treatment, leptin showed an anti-inflammatory phenotype after mechanical stress.

Effect of Bacterial Infection on Ghrelin Receptor Regulation in Periodontal Cells and Tissues.

The effect of bacterial infection on the expression of growth hormone secretagogue receptor (GHS-R) was investigated in periodontal cells and tissues, and the actions of ghrelin were evaluated. GHS-R was assessed in periodontal tissues of rats with and without periodontitis. Human gingival fibroblasts (HGFs) were exposed to Fusobacterium nucleatum in the presence and absence of ghrelin. GHS-R expression was determined by real-time PCR and immunocytochemistry. Furthermore, wound healing, cell viability, proliferation, and migration were evaluated. GHS-R expression was significantly higher at periodontitis sites as compared to healthy sites in rat tissues. F. nucleatum significantly increased the GHS-R expression and protein level in HGFs. Moreover, ghrelin significantly abrogated the stimulatory effects of F. nucleatum on CCL2 and IL-6 expressions in HGFs and did not affect cell viability and proliferation significantly. Ghrelin stimulated while F. nucleatum decreased wound closure, probably due to reduced cell migration. Our results show original evidence that bacterial infection upregulates GHS-R in rat periodontal tissues and HGFs. Moreover, our study shows that ghrelin inhibited the proinflammatory actions of F. nucleatum on HGFs without interfering with cell viability and proliferation, suggesting that ghrelin and its receptor may act as a protective molecule during bacterial infection on periodontal cells.

Effects of Obesity on Bone Healing in Rats.

Although the association between periodontitis and obesity is well explored, it is unclear whether obesity is associated with a worse therapeutic outcome after periodontal treatment. The aim of this study was to investigate the effects of obesity on bone healing with and without the application of regeneration-promoting molecules. A standardized bone fenestration-type defect was created over the root of the mandibular first molar in 15 Wistar rats. Ten animals received a high-fat, high-sucrose diet (HFSD), while the remaining five animals were fed a standard diet. During surgery, the fenestration defects from half of the HFSD-fed, i.e., obese animals, were treated with regeneration-promoting molecules (enamel matrix derivative; EMD). After four weeks, bone healing was evaluated by histomorphometry, TRAP staining and immunohistochemistry for RUNX2 and osteopontin. The analyses revealed that the spontaneous healing of the periodontal defects was compromised by obesity. Application of EMD partially compensated for the negative effect of obesity. Nevertheless, EMD-stimulated bone healing in obese animals was not better than the spontaneous healing in the obesity-free control group, indicating that obesity may also inhibit the stimulatory effects of regeneration-promoting molecules. Our results show that obesity can negatively influence bone healing and suggest that bone healing may be compromised in humans.

Filifactor alocis and Tumor Necrosis Factor-Alpha Stimulate Synthesis of Visfatin by Human Macrophages.

There is little known about the effect of the periodontopathogen Filifactor alocis on macrophages as key cells of the innate immune defense in the periodontium. Therefore, the aim of the present study was to investigate the effect of F. alocis and additionally of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) on visfatin and other pro-inflammatory and proteolytic molecules associated with periodontitis in human macrophages. The presence of macrophage markers CD14, CD86, CD68, and CD163 was examined in gingival biopsies from healthy individuals and periodontitis patients. Human macrophages were incubated with F. alocis and TNFα for up to 2 d. The effects of both stimulants on macrophages were determined by real-time PCR, ELISA, immunocytochemistry, and immunofluorescence. F. alocis was able to significantly stimulate the synthesis of visfatin by human macrophages using TLR2 and MAPK pathways. In addition to visfatin, F. alocis was also able to increase the synthesis of cyclooxygenase 2, TNFα, and matrix metalloproteinase 1. Like F. alocis, TNFα was also able to stimulate the production of these proinflammatory and proteolytic molecules. Our results highlight the pathogenetic role of F. alocis in periodontal diseases and also underline the involvement of visfatin in the aetiopathogenesis of periodontitis.

Regulation of Anti-Apoptotic SOD2 and BIRC3 in Periodontal Cells and Tissues.

The aim of the study was to clarify whether orthodontic forces and periodontitis interact with respect to the anti-apoptotic molecules superoxide dismutase 2 (SOD2) and baculoviral IAP repeat-containing protein 3 (BIRC3). SOD2, BIRC3, and the apoptotic markers caspases 3 (CASP3) and 9 (CASP9) were analyzed in gingiva from periodontally healthy and periodontitis subjects by real-time PCR and immunohistochemistry. SOD2 and BIRC3 were also studied in gingiva from rats with experimental periodontitis and/or orthodontic tooth movement. Additionally, SOD2 and BIRC3 levels were examined in human periodontal fibroblasts incubated with Fusobacterium nucleatum and/or subjected to mechanical forces. Gingiva from periodontitis patients showed significantly higher SOD2, BIRC3, CASP3, and CASP9 levels than periodontally healthy gingiva. SOD2 and BIRC3 expressions were also significantly increased in the gingiva from rats with experimental periodontitis, but the upregulation of both molecules was significantly diminished in the concomitant presence of orthodontic tooth movement. In vitro, SOD2 and BIRC3 levels were significantly increased by F. nucleatum, but this stimulatory effect was also significantly inhibited by mechanical forces. Our study suggests that SOD2 and BIRC3 are produced in periodontal infection as a protective mechanism against exaggerated apoptosis. In the concomitant presence of orthodontic forces, this protective anti-apoptotic mechanism may get lost.

Effects of obesity on periodontal tissue remodeling during orthodontic movement.

INTRODUCTION: Orthodontic movement triggers a sequence of cellular and molecular events that may be affected by different systemic conditions. This study evaluated the effect of obesity on rat periodontal tissue remodeling induced by mechanical orthodontic force. METHODS: Thirty-two Holtzman rats were distributed into 4 groups: control, obesity induction (O), orthodontic movement (M), and obesity induction and orthodontic movement (OM). Obesity was induced by a high-fat diet for 90 days. After 15 days of orthodontic movement, the animals were killed. Obesity induction was confirmed by animal body weight, adipose tissue weight, and serologic analysis. Periodontal tissue remodeling was evaluated using microcomputed tomography and histologic analysis. The gene expression of adipokines and cytokines in gingival tissues was evaluated. RESULTS: An increase in body and adipose tissue weight was observed in the obesity induction groups. The O group presented an increase in lipids and blood glucose. The OM group showed a decrease in bone volume fraction and bone mineral density compared with all other groups and a tendency for more rapid tooth movement than the M group. The OM group showed a higher quantity of inflammatory cells and higher Mmp1 expression than the O group. The O and OM groups showed higher Nampt expression than the control group and lower Nampt expression than the M group. CONCLUSIONS: Obesity modulates periodontal tissue remodeling during orthodontic movement and results in more inflammation and bone loss than in nonobese animals.

Regulation of Cyclooxygenase 2 by Filifactor alocis in Fibroblastic and Monocytic Cells.

Periodontitis is a prevalent chronic inflammatory disease triggered by a synergistic and dysbiotic microbiota present in the oral biofilm. This in vitro study is aimed at evaluating the regulation of cyclooxygenase (COX)2 expression and production by the periodontopathogen Filifactor alocis in human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells and also at investigating the underlying cellular pathway mechanisms. HGF-1 and THP-1 cells were exposed either to F. alocis or to the proinflammatory cytokine tumor necrosis factor alpha (TNFα) for 1 and 2 d to examine the COX2 expression by qPCR. COX2 protein levels were evaluated by ELISA in F. alocis-stimulated cells. Both types of cells were also stimulated with a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. F. alocis significantly (p < 0.05) increased COX2 at both transcriptional and protein levels in both HGF-1 and THP-1 cells. Moreover, the stimulatory effect of F. alocis on COX2 was more pronounced in HGF-1 cells in comparison to THP-1 cells. F. alocis upregulated the COX2 expression in a dose-dependent manner in both type cells at 1 d. TNFα also significantly (p < 0.05) increased the COX2 expression in both cells. After preincubation of HGF-1 and THP-1 cells either with a neutralizing anti-TLR2 antibody or with specific MAPK inhibitors, the F. alocis-upregulated COX2 expression was significantly (p < 0.05) suppressed at 1 d. Our in vitro study provides original evidence that F. alocis stimulates COX2 production in fibroblastic and monocytic cells through TLR2 and MAPK mechanisms, suggesting a role of this periodontopathogen in the etiopathogenesis of periodontitis.

Resistin Is Increased in Periodontal Cells and Tissues: In Vitro and In Vivo Studies.

Resistin, a proinflammatory adipokine, is elevated in many inflammatory diseases. However, little is known about its performance in periodontitis. The present study is aimed at evaluating resistin expression and synthesis in periodontal cells and tissues under inflammatory/microbial stress in addition to its effects on the periodontium. In vivo, 24 male rats were randomly divided into two groups: control and ligature-induced periodontal disease. After 6 and 12 days, animals were sacrificed to analyze gene expression of adipokines, bone loss, inflammation, and resistin synthesis. In vitro, human periodontal ligament (PDL) fibroblasts were used to evaluate the expression of resistin after inflammatory stimuli. In addition, PDL fibroblasts were exposed to resistin to evaluate its role on soft and hard tissue metabolism markers. The periodontitis group demonstrated significant bone loss, an increase in the number of inflammatory cells and vascular structures, an increase in resistin expression and synthesis, and a decrease in the expression of adiponectin, leptin, and its functional receptor. PDL fibroblasts showed a significant increase in resistin expression and synthesis in response to the inflammatory stimulus by IL-1ß. Resistin induced an increase in cytokine expression and a decrease in the regulation of some hard tissue and matrix formation genes in PDL fibroblasts. These data indicate that resistin is produced by periodontal cells and tissues, and this effect is enhanced by inflammatory stimuli. Moreover, resistin seems to interfere with soft and hard tissue metabolism during periodontitis by reducing markers related to matrix formation and bone tissue.

CXCL1, CCL2, and CCL5 modulation by microbial and biomechanical signals in periodontal cells and tissues-in vitro and in vivo studies.

OBJECTIVES: This study was established to investigate whether the chemokines CXCL1, CCL2, and CCL5 are produced in periodontal cells and tissues and, if so, whether their levels are regulated by microbial and/or mechanical signals. MATERIALS AND METHODS: The chemokine expression and protein levels in gingival biopsies from patients with and without periodontitis were analyzed by RT-PCR and immunohistochemistry. The chemokines were also analyzed in gingival biopsies from rats subjected to experimental periodontitis and/or orthodontic tooth movement. Additionally, chemokine levels were determined in periodontal fibroblasts exposed to the periodontopathogen Fusobacterium nucleatum and mechanical forces by RT-PCR and ELISA. RESULTS: Higher CXCL1, CCL2, and CCL5 levels were found in human and rat gingiva from sites of periodontitis as compared with periodontally healthy sites. In the rat experimental periodontitis model, the bacteria-induced upregulation of these chemokines was significantly counteracted by orthodontic forces. In vitro, F. nucleatum caused a significant upregulation of all chemokines at 1 day. When the cells were subjected simultaneously to F. nucleatum and mechanical forces, the upregulation of chemokines was significantly inhibited. The transcriptional findings were paralleled at protein level. CONCLUSIONS: This study provides original evidence in vitro and in vivo that the chemokines CXCL1, CCL2, and CCL5 are regulated by both microbial and mechanical signals in periodontal cells and tissues. Furthermore, our study revealed that biomechanical forces can counteract the stimulatory actions of F. nucleatum on these chemokines. CLINICAL RELEVANCE: Mechanical loading might aggravate periodontal infection by compromising the recruitment of immunoinflammatory cells.

Regulation of matrix metalloproteinase-1 by Filifactor alocis in human gingival and monocytic cells.

OBJECTIVES: Periodontitis is a highly prevalent chronic inflammatory disease caused by periodontopathogens, such as Filifactor alocis. This study sought to examine the matrix metalloproteinase (MMP)-1 synthesis by monocytic and fibroblastic cells in response to F. alocis and to unravel the underlying cellular mechanisms. MATERIAL AND METHODS: Gingival biopsies from periodontally healthy and periodontitis individuals were analyzed for the presence of F. alocis and MMP-1 by RT-PCR. Human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells were stimulated with F. alocis in the presence and absence of a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. MMP-1 expression and protein levels were studied by RT-PCR and ELISA, respectively. RESULTS: F. alocis was highly prevalent in biopsies from periodontitis patients but barely present in the healthy gingiva. Significantly higher MMP-1 expression levels were found in the inflamed gingiva as compared with healthy biopsies. F. alocis caused a significant and dose-dependent MMP-1 upregulation in both cells. The stimulatory effect of F. alocis on MMP-1 was TLR2- and MAPK-dependent and more pronounced on THP-1 cells as compared with HGF-1 cells. CONCLUSIONS: Our results demonstrate that F. alocis and MMP-1 are more prevalent at periodontitis sites. Additionally, our study provides original evidence that F. alocis can stimulate MMP-1 production by fibroblastic and monocytic cells, suggesting that F. alocis may contribute to periodontal breakdown through MMP-1. CLINICAL RELEVANCE: F. alocis and MMP-1 are linked to each other and key players in periodontitis, which may have significant implications for future diagnostic and treatment strategies.

Regulation of tyrosine hydroxylase in periodontal fibroblasts and tissues by obesity-associated stimuli.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamines and has been connected to aggravated progression of periodontal disease under chronic stress. Obesity is known to increase the risk of periodontitis and adipokines have been suggested to be a pathomechanistic link. This study examines if obesity-associated stimuli have regulatory effects on TH levels in periodontal cells and tissues. Human periodontal ligament fibroblasts were cultured in the presence of leptin or visfatin for up to 2 days. Untreated cells served as control. TH regulation was analyzed by real-time PCR, immunocytochemistry and ELISA. TH gene expression in periodontal tissues of normal-weight and obese rodents was determined. Examination of gingival biopsies from rats and patients with and without periodontal disease was performed by real-time PCR or immunohistochemistry. For statistics, ANOVA and post hoc tests were applied (p < 0.05). In vitro, TH gene expression and protein levels were increased by leptin and visfatin. In vivo, TH gene expression was upregulated in periodontal tissues of obese rodents as compared to normal-weight animals. Additionally, increased TH gene expression was found in rat gingival biopsies with experimental periodontitis. Human gingival biopsies from sites of periodontitis confirmed the animal data by demonstrating elevated TH levels at periodontally diseased sites. This study provides original evidence that obesity-associated stimuli induce a TH upregulation in periodontal cells and tissues. Since TH levels were also increased at periodontitis sites, our in vitro and animal findings suggest that this enzyme could represent a pathomechanism whereby obesity contributes to periodontitis.

In vivo and in vitro anti-inflammatory and pro-osteogenic effects of citrus cystatin CsinCPI-2.

Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (p < 0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (p < 0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.

Physicochemical, morphological, and biological analyses of Ti-15Mo alloy surface modified by laser beam irradiation.

Perform a physicochemical and morphological characterization of a Ti-15Mo alloy surface modified by laser beam irradiation and to evaluate in vitro the morphological response and proliferation of osteoblastic cells seeded onto this alloy. Disks were made of two different metals, Ti-15Mo alloy and cpTi, used as control. A total of four groups were evaluated: polished cpTi (cpTi-pol), laser-irradiated cpTi (cpTi-L), polished Ti-15Mo alloy (Ti-15Mo-pol), and laser-irradiated Ti-15Mo alloy (Ti-15Mo-L). Before and after laser irradiation of the surfaces, physicochemical and morphological analyses were performed: scanning electron microscopy (FEG-SEM), energy-dispersive spectroscopy (EDX), and X-ray diffraction (XRD). The wettability of the samples was evaluated by contact angle measurement. Murine preosteoblastic cells MC3T3-E1 were cultured onto the experimental disks for cell proliferation, morphology, and spreading analyses. Laser groups presented irregular-shaped cavities on its surface and a typical microstructured surface with large depressions (FEG-SEM). The contact angle for both laser groups was 0°, whereas for the polished groups was ≈ 77 and ≈ 78 for cpTi-pol and Ti-15Mo-pol, respectively. Cell proliferation analysis demonstrated a higher metabolic activity in the laser groups (p < 0.05). From the fluorescence microscopy, Ti-15Mo-L surface seems to induce greater cellular differentiation compared to the cpTi-L surface. The preliminary biological in vitro analyses suggested possible advantages of laser surface treatment in the Ti-15Mo alloy regarding cell proliferation and maturation.